Ultraconserved yet informative for species delimitation: UCEs resolve long-standing systematic enigma in Central European bees Morgan Gueuning1, Juerg Frey2, and Christophe Praz1 1Universite de Neuchatel Institut de Biologie 2Agroscope Standort Wadenswil May 5, 2020 Abstract Accurate and testable species delimitation hypotheses are essential for measuring, surveying and managing biodiversity. Today, taxonomists often rely on mitochondrial DNA barcoding to complement morphological species delimitations. Although COI barcoding has largely proven successful in assisting identifications for most animal taxa, there are nevertheless numerous cases where mitochondrial barcodes do not necessarily reflect the species history. For instance, what is regarded as one single species can be associated with two distinct DNA barcodes, which can point either to cryptic diversity or to deep within-species mitochondrial divergences with no reproductive isolation. In contrast, two or more species can share barcodes, for instance due to mitochondrial introgression. These intrinsic limitations of mitochondrial DNA barcoding can only be addressed with nuclear genomic markers, which are expensive, labour intensive, poorly repeatable, and often require high-quality DNA. To overcome these limitations, we examined the use of ultraconserved nuclear genetic elements (UCEs) as a quick and robust genomic approach to address such problematic cases of species delimitation. This genomic method was assessed using six different bee species complexes suspected to harbour cryptic diversity, mitochondrial introgression, or mitochondrial paraphyly. The sequencing of UCEs recovered between 686 and 1860 homologous nuclear loci and provided explicit species delimitation hypotheses in all investigated species complexes. These results provide strong evidence for the suitability of UCEs as a fast method for species delimitation even in recently diverged lineages. Furthermore, this study provided the first conclusive evidence for both mitochondrial introgression among distinct species, and mitochondrial paraphyly within a single bee species. 1. Introduction Given the unprecedented levels of biodiversity losses currently observed (Barnosky et al., 2011; Pievani, 2014, uncovering cryptic diversity and providing testable species hypotheses is an urgent task for taxonomists, especially for the hyperdiverse group of the insects (Hallmann et al., 2017; S´anchez-Bayo & Wyckhuys, 2019; Seibold et al., 2019). Traditionally, species were described by examining variation in morphological traits (Padial, Miralles, De la Riva, & Vences, 2010). They were delimited in a way to minimize within-species variation and to maximise between-species variation in sets of variable characters. However, morphological taxonomy is often challenged by a lack of variation between taxa, or conversely by sexual or generational polymorphisms within species. Both of which will lead to an absence of a “morphological” gap between species and may result in substantial levels of cryptic diversity (Karanovic, Djurakic, & Eberhard, 2016). To complement morphology, DNA barcoding was introduced as a reliable, fast, and cheap identification method (Brunner, Fleming, & Frey, 2002; Hebert, Cywinska, Ball, & DeWaard, 2003), and has since been extensively used not only for specimen identification but also for species delimitation. For insects, the 5’-region of the cytochrome oxidase subunit I (COI) gene has quickly become the DNA barcode gold standard due to the fact that, for many species, it demonstrated only very limited intra-species variation (i.e. generally below Posted on Authorea 17 Mar 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158447712.23176502 | This a preprint and has not been peer reviewed. Data may be preliminary. 1 3%) yet distinct differentiation between species (e.g., Brunner et al., 2002; Hebert et al., 2003; Meyer & Paulay, 2005). In combination with morphological identification, COI-barcoding was shown to be a powerful tool for species delimitation in bees (Pauly, No¨el,Sonet, Notton, & Boev´e,2019; Praz, M¨uller,& Genoud, 2019; Schmidt, Schmid-Egger, Morini`ere,Haszprunar, & Hebert, 2015). There are, nevertheless, numerous examples where COI-barcoding leads to an erroneous signal. A number of possible reasons for such problematic barcoding results have recently emerged. For example, a growing body of literature is reporting that mitochondrial inheritance is more complicated than initially thought, with rare cases of paternal leakage, heteroplasmy or recombination (Ladoukakis & Zouros, 2017; White, Wolff, Pierson, & Gemmell, 2008). Furthermore, mitochondrial genomes can be subject to evolutionary forces acting solely at the organelle level [e.g. mitochondrial introgression, Wolbachia infection or sex-biased asymmetries; (Toews & Brelsford, 2012)]. Although these events are generally considered rare (but see Klopfstein, Kropf, & Baur, 2016; Neumeyer, Baur, Guex, & Praz, 2014; Nichols, Jordan, Jamie, Cant, & Hoffman, 2012), they can considerably skew phylogenies or biodiversity estimates (Andriollo, Naciri, & Ruedi, 2015; Hinojosa et al., 2019; Mutanen et al., 2016). Consequently, species delimitation should rely on multiple sources of information (Carstens, Pelletier, Reid, & Satler, 2013) and for molecular markers, species delimitation should use genes of both mitochondrial and nuclear origin (Dupuis, Roe, & Sperling, 2012). In contrast to the uncommon suitability of COI as a species marker, the quest for similarly well-suited, universal and robust nuclear markers was so far unsuccessful. Several types of nuclear markers have been explored, but current candidates are all associated with serious drawbacks. For instance, single-copy nuclear genes (i.e. elongation factor 1 alpha [EF-1a] or 28S) or multicopy ribosomal DNA markers (i.e. internal transcribed spacer [ITS]) were explored (Leneveu, Chichvarkhin, & Wahlberg, 2009; Martinet et al., 2018; Soltani, B´enon,Alvarez, & Praz, 2017; Williams, Lelej, & Thaochan, 2019). However, the usefulness of these nuclear markers is often limited by the lack of phylogenetic resolution (Dellicour & Flot, 2018), and in insects, by within-genome variation of the multi-copy ribosomal genes (e.g. ITS), which is a major impediment to the sequencing workflow. For increased resolution, some studies have used population genetic markers such as microsatellites. Although microsatellites provide ample resolution for species delimitation (McKendrick et al., 2017), one major limitation is that loci are clade-specific and therefore require clade specific primers that have to be designed base on available genomic information. Alternative approaches that are slightly more universal and provide equal or higher resolution include genomic-reduction techniques such as RAD- or ddRAD sequencing (Lemopoulos et al., 2019). These methods are very powerful and can provide high- resolution, intraspecific information on population dynamics. However, as for microsatellites, RAD-seq is hampered by cost, workload and/or amount of high-quality DNA required. More importantly, datasets obtained from different studies and/or taxa are hardly joinable due to the lack of repeatability. This last limitation of RAD-sequencing is a severe drawback for species delimitation, since taxonomic work essentially builds upon previous hypotheses, with new data continuously complementing earlier datasets. Ideally, molecular species-delimitation should be based on: (i) nuclear and mitochondrial markers, to reflect gene flow of both nuclear and mitochondrial genes; (ii) genomic scale for nuclear markers to cover numerous independent loci; (iii) sufficiently variable to capture recently diverged species; (iv) repeatable, so that datasets can be complemented once more material is available; (v) universal to the extent that datasets can complement each other. In 2012, ultraconserved elements (UCEs) were introduced as a quick and essentially universal way to obtain “thousands of genetic markers spanning multiple evolutionary timescales” (Faircloth et al., 2012). UCEs appear to fulfil many of the above mentioned requirements for nuclear markers. However, whether they harbor enough variation to capture divergence among recently diverged species remains an open question, since by definition they are highly conserved. In this study, we examine the use of UCEs for species delimitation in Central European bees. We include examples of both putative mitochondrial introgression and of multiple “barcodes” per species, investiga- ting how UCEs can overcome the main drawbacks for species delimitation using DNA barcoding developed above. We focused on the following European species complexes: Andrena amieti/allosa/bicolor/montana ; Andrena barbareae/cineraria; A. dorsata/propinqua; A. carantonica/trimmerana/rosae; Lasioglossum al- Posted on Authorea 17 Mar 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158447712.23176502 | This a preprint and has not been peer reviewed. Data may be preliminary. 2 pigenum/bavaricum/cupromicans; Nomada goodeniana/succincta . Mitochondrial introgressions have been suggested for four of these cases (Schmidt et al., 2015; see details below); low-divergence were suggested for the controversial A. carantonica /trimmeranacomplex (Schmidt et al. 2015); while deep within-species divergences not associated with morphological differentiation have been documented theAndrena amie- ti/allosa/bicolor/montana clade (Praz et al. 2019). Most of these cases are also controversial with respect to morphological delimitations, so that